Pseudocercospora fijiensis causes black Sigatoka disease of banana. It is one of the most damaging threats of the crop requiring excessive fungicide applications for disease control as the major export “Cavendish” clones are highly susceptible. The consequence of this practice is the reduced efficacy of disease management strategies due to increasing levels of fungicide resistance. In this thesis the history and current practices of black Sigatoka disease management as well as the underlying mechanisms of fungicide resistance to a major group of fungicides are described. We discovered that both target site mutations and promotor insertions are crucial for modulating sensitivity. The more insertions, the higher the expression of the gene and the more resistant the strain. Using this information, we advocate modern monitoring techniques and improved disease control strategies as well as the urgent need for innovative banana breeding to develop resistant varieties for a sustainable global banana production.

Resistance to antimicrobial agents is a serious problem for both medicine and agriculture. The initial success of such toxins is due to absence of resistant genotypes in pathogen populations before treatment. The initial low frequency of resistance may be explained by negative pleiotropic effects of the resistance mutations on fitness in the absence of the toxin. However, when resistant and sensitive natural isolates are compared, no cost of resistance may often be seen. This high fitness of resistant genotypes is explained by the existence of compensatory mutations, i.e. mutations that ameliorate the negative effect of the initial resistance mutation. The accumulation of these compensatory mutations can only occur at a sufficient rate if the population of resistant genotypes is large, which in part depends on the pesticide use. As a result, resistance may be stably maintained in a population after pesticide treatment, whereas it was absent before pesticide treatment. Resistance to the phenylamide pesticide metalaxyl is common in populations of Phytophthora infestans, the oomycete that causes the late blight disease in potato. This resistance evolved within a few years after metalaxyl became commercially available in 1977. Such fast evolution of pesticide resistance may present a serious threat for potato production. However, fast evolution of resistance to other pesticides has not been described in P. infestans, whereas resistance to metalaxyl and other pesticides are common in other oomycetes. In the absence of metalaxyl, resistant isolates of P. infestans have a higher fitness during epidemics compared to sensitive isolates. This observation has led to the assumption that metalaxyl resistance has a direct positive effect on epidemic fitness. A reduction of the survival during the winter has been put forward as an explanation for the absence of resistance before the commercial release of metalaxyl. However, given a high mutation rate towards metalaxyl resistance, a direct positive effect of metalaxyl resistance would always lead to a high frequency of resistance, even in the absence of metalaxyl. In this thesis, we report on an experimental evolution approach to investigate the fast evolution and apparent stability of metalaxyl resistance in P. infestans. We used an experimental evolution procedure because it has several advantages. First, it enables a detailed inspection of the trajectory of evolutionary changes (e.g. in resistance phenotype). Second, it enables comparisons of fitness components between near-isogenic genotypes that only differ in their sensitivity to metalaxyl, whereas conclusions based on comparisons of field isolates and offspring from sexual crosses are hampered by differences in the genetic background. Two experiments were performed to investigate the rate of evolution of resistance to a number of pesticides. First, we used a fluctuation test to estimate the mutation rate towards resistance against metalaxyl, fluazinam and cyazofamid. This fluctuation test, however, was not suitable to estimate the mutation rate in P. infestans, due to background growth of sensitive genotypes for metalaxyl and due to the low amount of zoospores that can be obtained from a single colony for the other two pesticides. Second, we analyzed the response of selection on low pesticide concentrations. Serial transfer of zoospores on a constant low concentration of metalaxyl resulted in full resistance. The same procedure on a low concentration of cyazofamid with a similar effect on growth rate, however, did not result in full resistance. The genotypes that emerged after the selection procedure were compared with the sensitive ancestor with respect to a number of fitness components in the absence of metalaxyl. This approach did not reveal a cost of resistance. On the contrary, resistant genotypes showed a higher in vitro fitness compared to their sensitive ancestor. However, the results from a population survey during a nation-wide ban of metalaxyl containing pesticides in the Netherlands were suggestive of a negative effect of resistance on winter survival and on epidemic fitness. These results suggest that the relative high frequency of metalaxyl resistant isolates on untreated fields can only be explained by assuming that metalaxyl resistance has an initial cost that is followed by compensatory mutations that only restores epidemic fitness. A new selection procedure that included serial transfer of zoospores, revealed a small initial cost on in vitro performance by increasing the lag phase before the start of growth. Subsequent compensatory evolution under the selection conditions could then explain the absence of a cost of resistance in previous experiments. However, no such compensatory evolution was directly observed in this latter selection procedure. The results presented in this thesis suggest that metalaxyl resistance is selected on a wide range of concentrations, whereas this is not the case for (some) other pesticides. This may explain the relative fast evolution of resistance to metalaxyl relative to that for other pesticides. Additionally, the absence of resistance before the commercial release of metalaxyl can be explained by a negative effect due to delaying growth. Finally, the methodology of experimental evolution may be a valuable addition to the multitude of methods used to study the population dynamics of this important pathogen.

ATP-binding cassette (ABC) transporters are membrane proteins that utilise the energy derived from the hydrolysis of ATP to drive the transport of compounds over biological membranes. They are members of one of the largest protein families to date, present in both pro- and eukaryotic organisms. ABC transporters play an essential role in multidrug resistance (MDR) of cancer cells to chemically unrelated compounds. ABC transporters involved in drug resistance have also been described in filamentous fungi. In plant pathogenic fungi ABC transporters may act as virulence factors if they mediate secretion of host-specific toxins efence compounds during pathogenesis. Such a role in pathogenesis has been demonstrated for the ABC transporters ABC1 from Magnaporthe grisea , BcatrB from Botrytis cinerea , and GpABC1 from Gibberella pulicaris .

In our laboratory ABC transporters from Mycosphaerella graminicola (Fückel) Schröter (anamorph state: Septoria tritici Rob.ex.Desm.), the causal agent of septoria tritici blotch of wheat, are studied. This disease can cause a significant reduction in yield. Typical disease symptoms are necrotic spots filled with the asexual pycnidia and sexual pseudothecia of the fungus. Formation of the necrotic lesions may be associated with secretion of phytotoxic compounds by the pathogen. On the other hand, wheat is known to produce plant defence compounds, such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) and fluorescent compounds produced around infected stomata. Therefore, the fungus may have evolved specific ABC transporters that secrete toxins, or reduce the intracellular accumulation of plant defence compounds. Disease management of M. graminicola has widely involved the use of azole fungicides such as cyproconazole, propiconazole, and tebuconazole. The mode of action of these fungicides is based on inhibition of cytochrome P450 sterol 14α-demethylase (P45014DM ) activity, a key enzyme in the sterol biosynthetic pathway. In plant pathogenic fungi four major mechanisms of resistance to azoles have been reported. One of these is reduced accumulation of the fungicides in mycelium, attributed to an energy-dependent efflux mechanism mediated by ABC transporters. Other possible resistance mechanisms include mutations in the CYP51 gene encoding P450 14DM as well as overexpression of this gene.

Recently, Zwiers and De Waard (2000) cloned and characterised the ABC transporter genes MgAtr1 and MgAtr2 from M. graminicola . Research in the current study was primarily focused on cloning additional ABC transporter genes from the plant pathogenic fungus M.graminicola and examining their physiological role during pathogenesis and in providing protection of the fungus against natural and synthetic toxic compounds. In addition, we have also studied the resistance mechanisms to azole fungicides in M. graminicola that might be operating in laboratory-generated azole-resistant mutants and field isolates of this fungus with different sensitivity levels to these compounds. ABC transporters could have an important role in MDR resistance development. Ways to overcome such problems by inhibiting the function of these proteins with compounds able to modulate their function were also investigated. Such knowledge could be of great importance in disease control management of this fungus and lead to new and innovative disease control methods.

Chapter 1 describes M. graminicola and its importance in agriculture. In addition, mechanisms of resistance to azole fungicides are presented.

Chapter 2 comprises a review, describing the function of ABC transporters from filamentous fungi in pathogenesis and protection against natural and synthetic toxic compounds. Members of the major facilitator superfamily (MFS) of membrane transporters from filamentous fungi are also described, since these proteins can mediate similar functions in cells as ABC transporters.

Chapter 3 describes the cloning and characterisation of the ABC transporter genes MgAtr3, MgAtr4, and MgAtr5 using a PCR-based approach. Sequence analysis showed that the encoded proteins exhibit a topology similar to that of MgAtr1 and MgAtr2 from M. graminicola . Northern analysis demonstrated that the genes display distinct but overlapping expression profiles when treated with a number of natural or synthetic toxic compounds known to be either inducers or substrates of ABC transporters.

In Chapter 4 the role in MDR of MgAtr1-MgAtr5 is studied. This was done by complementation of Saccharomyces cerevisiae mutants with the M. graminicola ABC transporter genes and by analysis of ABC transporter disruption or replacement mutants of M. graminicola with respect to sensitivity to natural and synthetic toxic compounds as well as antagonistic bacteria. Results indicate that ABC transporters from M. graminicola can play a role in protection of the fungus against natural and synthetic toxic compounds.

In Chapter 5 the role of MgAtr1-MgAtr5 as virulence factors during pathogenesis on wheat seedlings is studied. Disruption or replacement strains of MgAtr1, MgAtr2, MgAtr3, and MgAtr5 displayed an unaltered phenotype in comparison to the wild-type control but virulence of MgAtr4 disruption mutants was significantly reduced on seedlings of all wheat cultivars tested. Therefore, MgAtr4 is a virulence factor of M. graminicola during pathogenesis on wheat. This is the first virulence factor identified so far from this important plant pathogen.

Chapter 6 describes studies on mechanisms of resistance to azole fungicides in azole-resistant laboratory-generated mutants of M. graminicola . These include efflux mechanisms mediated by ABC transporters, overexpression of CYP51, and mutations in the coding sequence of this gene. The results indicate that multiple mechanisms may be responsible for reduced sensitivity of the mutants to azoles.

Chapter 7 describes molecular mechanisms that account for variation in base-line sensitivity to azole fungicides in field isolates of M. graminicola and hence, complement results described in Chapter 6 for the field situation. Genetic analysis showed that azole sensitivity in M. graminicola is a polygenic trait. Overexpression of ABC transporter genes and CYP51 may explain the reduced azole sensitivity of some field isolates, indicating that multiple mechanisms could account for differences in base-line sensitivity to azoles.

In Chapter 8 the antimicrobial activity of the azole fungicides cyproconazole and propiconazole alone and in combination with ABC transporter modulators against M. graminicola is studied. Interactions in the mixtures are tested using the Colby and Wadley method with a wild-type M. graminicola isolate that showed moderate sensitivity to azole fungicides. Analysis with both methods showed that interactions between the compounds in most combinations tested are additive.

Chapter 9 presents a summarising discussion of the thesis.

In conclusion, data presented in this thesis show that ABC transporters from M. graminicola have a number of important functions. They can act as virulence factors of plant pathogens. In addition, they may provide protection against natural and synthetic, toxic compounds and account for base-line sensitivity and fungicide resistance of fungi to azole fungicides.